Gelsekoumidines A and B: Two Pairs of Atropisomeric Bisindole Alkaloids from the Roots of Gelsemium elegans

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1 Gelsekoumidines A and B: Two Pairs of Atropisomeric Bisindole Alkaloids from the Roots of Gelsemium elegans Wei Zhang a,b, Wei Xu a, Gui-Yang Wang a, Xue-Ying Gong a, Ni-Ping Li a, Lei Wang a,b,*, Wen-Cai Ye a,b, a Institute of Traditional Chinese Medicine & Natural Products, and JNU-HKUST Joint Laboratory for Neuroscience & Innovative Drug Research, College of Pharmacy, Jinan University, Guangzhou , People s Republic of China b Guangdong Province Key Laboratory of Pharmacodynamic Constituents of TCM and New Drugs Research, Jinan University, Guangzhou , People s Republic of China 1

2 Contents 1. General experimental procedures Plant material Extraction and isolation of Physico-chemical constants of X-Ray crystallographic analysis of 1a Dynamic HPLC analyses of 1a and 1b Quantum chemical ECD calculation method Bioassay methods Figure S5. The UV of compound 1 in MeOH Figure S6. The IR (KBr disc) of compound Figure S7. The HR-ESI-MS of compound Figure S8. The 1 H NMR spectrum for compound 1 in CD 3 OD Figure S9. The 13 C NMR spectrum of compound 1 in CD 3 OD Figure S10. The DEPT-135 spectrum of compound 1 in CD 3 OD Figure S11. The 1 H- 1 H COSY spectrum of compound 1 in CD 3 OD Figure S12. The HSQC NMR spectrum of compound 1 in CD 3 OD Figure S13. The HMBC spectrum of compound 1 in CD 3 OD Figure S14. The NOESY spectrum of compound 1 in CD 3 OD Figure S15. 1 H NMR spectra for compound 1 in CDCl 3, DMSO-d 6, CDOD 3, and pyridine-d Figure S16. The UV of compound 2 in MeOH Figure S17. The IR (KBr disc) of compound Figure S18. The HR-ESI-MS of compound Figure S19. The 1 H NMR spectrum for compound 2 in CD 3 OD Figure S20. The 13 C NMR spectrum of compound 2 in CD 3 OD Figure S21. The DEPT-135 spectrum of compound 2 in CD 3 OD Figure S22. The 1 H- 1 H COSY spectrum of compound 2 in CD 3 OD Figure S23. The HSQC spectrum of compound 2 in CD 3 OD Figure S24. The HMBC spectrum of compound 2 in CD 3 OD Figure S25. The NOESY spectrum of compound 2 in CD 3 OD

3 1. General experimental procedure Melting points were obtained on an X-5 micro melting point apparatus without correction (Fukai Instrument, Beijing, China). Optical rotations were determined using a JASCO P-1020 digital polarimeter (Jasco, Tokyo, Japan) at 25 C. UV spectra were recorded on a Jasco V-550 UV/VIS spectrophotometer (Jasco, Tokyo, Japan). IR spectra were determined on a Jasco FT/IR-480 plus Fourier transform infrared spectrometer (Jasco, Tokyo, Japan) using KBr pellets. ECD spectra were obtained on a Jasco J-810 spectropolarimeter (Jasco, Tokyo, Japan) at room temperature. HR-ESI- MS was carried out on Agilent 6210 LC/MSD TOF mass spectrometer (Agilent Technologies, CA, USA). NMR spectra were measured on Bruker AV-400 and AV- 500 spectrometers (Bruker, Switzerland) with TMS as an internal standard. Singlecrystal data were performed using Oxford-Diffraction SuperNova diffractometer and Cu K radiation. Column chromatography (CC) were performed on Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), silica gel ( mesh; Qingdao Marine Chemical Inc., Qingdao, P. R. China), and ODS (YMC, Kyoto, Japan). Preparative HPLC was performed on an Agilent 1260 Chromatograph equipped with a G1311C pump and a G1315D photodiode array detector (Agilent Technologies, CA, USA) with a Waters Xbridge C 18 OBD reversed-phase column ( mm, 5 mm, USA). All solvents used in HPLC were of chromatographic grade (Fisher Scientific, New Jersey, USA). 2. Plant material The roots of Gelsemium elegans were collected from Conghua city, Guangdong province of P. R. China, in October of A voucher specimen (No ) identified by Prof. Guang-Xiong Zhou (Jinan University) was deposited in the Institute of Traditional Chinese Medicine & Natural Products, Jinan University, Guangzhou, P. R. China. 3. Extraction and isolation of 1-2 The air-dried G. elegans (18 kg) were pulverized and extracted with 95% EtOH. The extract (1.7 kg) was suspended in H 2 O and acidified with 5% HCl to ph 3. The acidic suspension was partitioned with CHCl 3 to remove the neutral components. 3

4 Then the aqueous layer was basified with 10% aqueous ammonia to ph 9 and extracted with CHCl 3 to obtain a total alkaloid fraction. The alkaloid fraction (220 g) was subjected to silica gel column chromatography using CHCl 3 -MeOH (100:0 0:100, v/v) as gradient eluent to afford eleven fractions (Frs. A-K). Fr. D (16.5 g) was separated by ODS CC with MeOH-H 2 O (1:9 to 10:0, v/v) to yield six fractions (Frs. D1-D6). Fr. D2 (3.2 g) was chromatographed on Sephadex LH-20 (CHCl 3 -MeOH, 1:1, v/v) and then was purified by preparative HPLC (ACN-H 2 O, 40:60, v/v) to afford compounds 1 (36.6 mg) and 2 (15.4 mg). 4. Physico-chemical constants of 1-2 Compound 1: light-yellow crystals (MeOH-H 2 O); mp ; [ ] 25 D = (c = 0.50, CHCl 3 ); 1 H and 13 C NMR data, see Table 1; UV (MeOH) λ max (log ε): 208 (3.15), 272 (2.97), 302 (2.27) nm; IR (KBr) ν max : 3285, 2925, 1719, 1643, 1588, 1466, 1322, 1109, 866, 754, 682 cm -1 ; HR-ESI-MS: m/z [M+H] + (calcd for C 39 H 41 N 4 O 6, ); ECD (MeCN) λ max (Δε) 236 (+ 8.3), 268 (+ 23.0), 217 (- 12.0), 299 (- 40.6). Compound 2: amorphous powder; [ ] 25 D = (c = 0.50, CHCl 3 ); 1 H and 13 C NMR data, see Table S1; UV (MeOH) λ max (log ε): 208 (3.28), 266 (2.28), 306 (2.95) nm;ir (KBr) ν max : 3434, 2935, 1725, 1618, 1470, 1326, 1207, 1118, 1062, 946, 757, 624 cm -1 ; HR-ESI-MS: m/z [M+H] + (calcd for C 39 H 41 N 4 O 5, ); ECD (MeCN) λ max (Δε) 228 (+ 17.5), 267 (+ 15.5), 299 (- 46.3). Table S1. 1 H (500 MHz) and 13 C NMR (125 MHz) Data of 2 in CD 3 OD ( in ppm, J in Hz) No. 2b 2a a) H C a) H C br s br s d (8.4) d (8.4) d (8.4) d (8.4)

5 ddd br d (7.6) br d (7.6) d (4.7) d (4.7) d (11.5) d (11.5) d (11.5) d (11.5) s s s s ' ' ' d (8.6) ' ' ' ' ' 7.11 ddd (7.7, 7.7, 0.9) ddd (7.7, 7.7, 0.9) ' 7.30 ddd (7.7, 7.7, 0.9) ddd (7.7, 7.7, 0.9) ' 6.95 br d (7.7) br d (7.7) ' ' ' ' ' ' 1.92 s s ' ' ' 3.93 s s 64.0 a) Overlapped signals were reported without designating multiplicity. 5

6 5. X-Ray Crystallographic analysis of 1a Crytallographic data for 1a have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 IEZ, UK (fax: +44-(0) or Table S2. Crystal data and structure refinement for 1a Empirical formula C 39 H 40 N 4 O 6 Formula weight Temperature Wavelength Crystal system (10) K Å Monoclinic Space group P Unit cell dimensions a = (3) Å = 90 b = (4) Å = (17) c = (3) Å = 90 Volume (12) Å 3 Z 4 Density (calculated) Mg/m 3 Absorption coefficient mm -1 F(000)

7 Crystal size mm 3 Theta range for data collection to Index ranges -15<=h<=14, -25<=k<=24, -17<=l<=17 Reflections collected Independent reflections [R(int) = ] Completeness to theta = % Absorption correction Semi-empirical from equivalents Max. and min. transmission and Refinement method Full-matrix least-squares on F 2 Data / restraints / parameters 13180/1/963 Goodness-of-fit on F Final R indices [I>2sigma(I)] R 1 = , wr 2 = R indices (all data) R 1 = , wr 2 = Absolute structure parameter 0.07 (15) Extinction coefficient n/a Largest diff. peak and hole and e.å Dynamic HPLC analyses of 1a and 1b The isomers 1a and 1b isolated by HPLC were respectively analyzed at different time (0 h, 0.5 h, 1 h, and 24 h; room temperature). The HPLC/UV spectra were obtained by an Agilent 1260 instrument equipped with a DAD detector (Agilent 1260, DADVL, USA) and a Waters Xbridge C 18 OBD ( mm, 5 μm). The column temperature was controlled at 298 K by an Agilent 1260 TCCVL (USA).The mobile phase was ACN-H 2 O (40:60, v/v) (λ = 210 nm; flow rate: 1 ml/min). 7

8 Figure S1. Dynamic HPLC spectra of 1a and 1b 7. Quantum chemical ECD calculation method Computational data of 1 Figure S2. Calculated and experimental CD spectra of 1 8

9 The systematic random conformational analysis of the enantiomers of compound 1 was performed in the SYBYL 8.1 program by using MMFF94s molecular force field, which afforded 32 conformers of 1, with an energy cutoff of 10 kcal mol -1 to the global minima. All the obtained conformers were further optimized using DFT at the B3LYP/6-31+G(d) level in gas phase by using Gaussian09 software, [1] and 30 conformers of 1 were selected. All of the optimized stable conformers were used for TDDFT computation of the excited stats at the same levels, with the consideration of the first 30 excitations. The overall ECD curves of 1 were weighted by Boltzmann distribution of each conformer (with a half-bandwidth of 0.3 ev), with a UV correction of 10 nm. The calculated ECD spectra of 1 were subsequently compared with the experimental one. The ECD spectra were produced by SpecDis 1.6 software. [2] Figure S3. Key molecular orbitals involved in important transitions regarding the ECD spectra of conformer 25 in the gas phase at the B3LYP/6-31+G(d) level. 9

10 Table S3. Key transitions and their related rotatory and oscillator strengths of conformer 25 of 1 at the B3LYP/6-31+G(d) level in the gas phase. HOMO is 175 No. Energy (cm -1 ) Wavelength (nm) R (length) Osc. Strength Major contribs HOMO->LUMO (79%), HOMO- >L+1 (18%) H-1->LUMO (88%) H-2->LUMO (22%), HOMO- >LUMO (11%), HOMO->L+1 (58%) H-4->LUMO (37%), H-3->LUMO (13%), H-2->LUMO (31%) H-4->LUMO (24%), H-2->LUMO (45%), HOMO->L+1 (11%) HOMO->L+2 (95%) H-6->LUMO (11%), H-4->LUMO (10%), H-3->LUMO (53%) H-1->L+2 (80%) H-5->LUMO (14%), HOMO->L+4 (65%) H-6->LUMO (18%), H-5->LUMO (29%), H-4->LUMO (12%), H-3- >LUMO (19%) H-6->LUMO (21%), H-5->LUMO (24%), HOMO->L+4 (31%) H-1->L+1 (89%) HOMO->L+3 (88%) H-2->L+1 (90%) H-9->LUMO (28%), H-7->LUMO (23%), H-1->L+3 (21%) H-9->LUMO (15%), H-1->L+3 (45%) H-5->LUMO (20%), H-5->L+1 (28%), H-3->L+1 (23%), H-2->L+4 (18%) 10

11 HOMO->L+5 (88%) H-9->LUMO (21%), H-8->LUMO (42%), H-6->LUMO (13%) H-9->LUMO (25%), H-8->LUMO (13%), H-7->LUMO (39%) H-12->LUMO (13%), H-8->LUMO (40%), H-6->L+1 (11%) HOMO->L+6 (92%) H-9->L+2 (56%), H-9->L+3 (10%) H-12->LUMO (12%), H-4->L+1 (10%), H-3->L+1 (36%) HOMO->L+7 (79%) H-10->LUMO (22%), H-6->L+1 (24%), H-5->L+1 (10%), H-4->L+1 (24%) H-4->L+1 (49%), H-3->L+1 (20%) H-2->L+2 (98%) H-1->L+5 (54%) H-12->LUMO (23%), H-10->LUMO (40%) Table S4. Z-matrices and Cartesian coordinates of conformer 25 of 1 Row Tag Symbol Bond Angle Dihedral X Y Z 1 1 C C C C C C N C C C O C C C C

12 16 16 C C N C C H O H H H O C C C C C C C N C C C C C C C C O C N C C C

13 49 49 C C O H O C H H H H H H H H H H H H H H H H H H H H H H H H H H H

14 82 82 H H H H H H H H Computational data of 2 The systematic random conformational analysis of the enantiomers of compound 1 was performed in the SYBYL 8.1 program by using MMFF94s molecular force field, which afforded 28 conformers of 1, with an energy cutoff of 10 kcal mol -1 to the global minima. All the obtained conformers were further optimized using DFT at the B3LYP/6-31+G(d) level in gas phase by using Gaussian09 software, [1] and 21 conformers of 1 were selected. All of the optimized stable conformers were used for TDDFT computation of the excited stats at the same levels, with the consideration of the first 30 excitations. The overall ECD curves of 1 were weighted by Boltzmann distribution of each conformer (with a half-bandwidth of 0.3 ev), with a UV correction of 10 nm. The calculated ECD spectra of 1 were subsequently compared with the experimental one. The ECD spectra were produced by SpecDis 1.6 software. [2] 14

15 Figure S4. Key molecular orbitals involved in important transitions regarding the ECD spectra of conformer 18 in the gas phase at the B3LYP/6-31+G(d) level. Table S5. Key transitions and their related rotatory and oscillator strengths of conformer 18 of 1 at the B3LYP/6-31+G(d) level in the gas phase. HOMO is 171 No. Energy (cm -1 ) Wavelength (nm) R (length) Osc. Strength Major contribs HOMO->LUMO (78%), HOMO- >L+1 (18%) H-1->LUMO (95%) H-2->LUMO (44%), HOMO->L+1 (46%) H-4->LUMO (20%), H-2->LUMO (32%), HOMO->L+1 (23%) H-4->LUMO (31%), H-3->LUMO (26%), H-2->LUMO (19%) H-5->LUMO (10%), H-4->LUMO 15

16 (17%), H-3->LUMO (42%) HOMO->L+2 (77%) H-1->L+1 (26%), H-1->L+2 (49%) H-7->LUMO (22%), H-5->LUMO (28%), HOMO->L+4 (17%) H-1->L+1 (67%), H-1->L+2 (22%) H-5->LUMO (19%), HOMO->L+3 (10%), HOMO->L+4 (56%) H-2->L+1 (73%) H-7->LUMO (25%), H-5->LUMO (22%), H-2->L+1 (17%) H-8->LUMO (13%), H-6->LUMO (15%), H-1->L+3 (12%), HOMO- >L+3 (29%) H-6->LUMO (12%), H-3->L+1 (24%), H-2->L+4 (10%), HOMO- >L+3 (22%) H-3->L+1 (16%), H-2->L+4 (10%), HOMO->L+3 (22%) H-8->LUMO (10%), H-1->L+3 (30%), HOMO->L+5 (31%) H-1->L+3 (17%), HOMO->L+5 (51%) H-10->LUMO (10%), H-8->LUMO (27%), H-6->LUMO (35%) H-11->LUMO (18%), H-11->L+1 (10%), H-7->LUMO (23%), H-7- >L+1 (17%), H-5->L+1 (10%) HOMO->L+6 (81%) H-10->LUMO (42%), H-9->LUMO (20%) H-9->LUMO (72%) H-8->LUMO (11%), H-8->L+2 (37%) H-4->L+1 (58%) H-11->LUMO (12%), H-10->LUMO 16

17 (16%), H-5->L+1 (13%), H-4->L+1 (10%), H-3->L+1 (22%) HOMO->L+7 (70%) H-1->L+3 (12%), H-1->L+4 (44%), H-1->L+5 (23%) H-1->L+4 (39%), H-1->L+5 (37%) H-2->L+2 (86%) Table S6. Z-matrices and Cartesian coordinates of conformer 18 of 2 Row Tag Symbol Bond Angle Dihedral X Y Z 1 1 C C C C C C N C C C O C C C C C C N C C H O H H

18 25 25 H C C C C C C C N C C C C C C C C O C N C C C C C H O O C H H H H H H

19 60 60 H H H H H H H H H H H H H H H H H H H H H H H H H H H H H Bioassay methods MTT assay method The cell viability of Macrophage RAW cells was performed by MTT assay. Cells were cultured in DMEM containing 10% FBS (V/V) with 19

20 penicillin (100 U/mL) and streptomycin (100 U/mL) at 37 C with 5% CO 2 (V/V). Cells at the logarithmic phase were seeded in 96-well plates at cells per well. After preincubation overnight, cells were treated with various concentrations of compounds and cultured for 24 h. Then, 40 μl of 5 mg/ml MTT solu-tions was added to each well and incubated for 4 h. After complete removal of the medium, 100 μl of DMSO was added to each well to dissolve the formazan crystals. And the optical density (OD) was rec-orded at 570 nm. Viability rate = the average OD value of the treatment group/the average OD value of the control group. Determination of LPS-Induced NO Production in Macrophage RAW Cells. The macrophage RAW cells were cultivated in DMEM containing 10% FBS (V/V) with penicillin (100 U/mL) and streptomycin (100 U/mL) at 37 C in a humidified atmosphere with 5% CO 2 (V/V). The cells were allowed to grow in 96-well plates with cells to treat test compounds. Cells were pre-incubated for 1 h in the absence or presence of compounds before the addition of LPS for 24 h. Then, the culture supernatant (100 μl) was incubated with a Griess reagent (100 μl, Sigma) at room temperature for 10 min. The absorbance was measured at 550 nm against a calibration curve with sodium nitrite standards. All the experiments were performed in three independent replicates. 20

21 Figure S5. The UV of compound 1 in MeOH Figure S6. The IR (KBr disc) of compound 1 Figure S7. The HR-ESI-MS of compound 1 21

22 Figure S8. 1 H NMR spectrum for compound 1 in CD 3 OD [1a (blue) and 1b (red)] Figure S9. The 13 C NMR spectrum of compound 1 in CD 3 OD Figure S10. The DEPT-135 spectrum of compound 1 in CD 3 OD 22

23 Figure S11. The 1 H- 1 H COSY spectrum of compound 1 in CD 3 OD Figure S12. The HSQC spectrum of compound 1 in CD 3 OD 23

24 Figure S13. The HMBC spectrum of compound 1 in CD 3 OD Figure S14. The NOESY spectrum of compound 1 in CD 3 OD 24

25 a b c d Figure. S15. 1 H NMR spectra for compound 1 in CDCl 3 (a), DMSO-d 6 (b), CDOD 3 (c), and pyridine-d 5 (d). 25

26 Figure S16. The UV of compound 2 in MeOH Figure S17. The IR (KBr disc) of compound 2 Figure S18. The HR-ESI-MS of compound 2 26

27 Figure S19. 1 H NMR spectrum for compound 2 in CD 3 OD [2a (blue) and 2b (red)] Figure S20 The 13 C NMR spectrum of compound 2 in CD 3 OD Figure S21. The DEPT-135 spectrum of compound 2 in CD 3 OD 27

28 Figure S22. The 1 H- 1 H COSY spectrum of compound 2 in CD 3 OD Figure S23. The HSQC spectrum of compound 2 in CD 3 OD 28

29 Figure S24. The HMBC spectrum of compound 2 in CD 3 OD Figure S25. The NOESY spectrum of compound 2 in CD 3 OD 29

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